Monitoring of Metabolic Disorders Through Electro‐Sensing of Riboflavin by Novel Super‐Conductive Silver Ink Modified Paper

ABSTRACT

Riboflavin (RIB), or vitamin B2, plays a key role in cellular protection against oxidative stress, and its deficiency can cause severe physiological complications, including metabolic disorders, photosensitivity, anaemia, growth delay and gastrointestinal problems. Rapid and accurate quantification of RIB in real biological samples is important. In this study, a novel paper‐based electrochemical microsensor was fabricated for RIB detection in blood plasma samples. Highly conductive silver ink was synthesized and loaded into a ballpoint pen, enabling direct drawing of microscale (∼85.78 µm) electrodes onto a cellulose substrate. Owing to the optimized ink formulation, the drawn electrodes dried immediately (∼3 s) at room temperature, producing uniform, well‐defined and highly conductive patterns without spreading. In the presence of induced stress conditions, the electrodes exhibited excellent thermal, mechanical and moisture stability, with resistances remaining stable at 1.5 Ω (at 150°C), 2.9 Ω (humidity exposure) and 2 Ω (mechanical bending). The three‐dimensional network formed by silver ink on photographic paper substrate preserved conductivity under stress and prevented structural cracking. RIB was detected using cyclic voltammetry, square‐wave voltammetry, chronoamperometry and differential pulse voltammetry. Under optimized conditions, the sensor showed a linear response over 0.4–10 µM with a lower limit of quantification of 0.4 µM. Intra‐day and inter‐day repeatability ranged from 1.47% to 6.35% and 3.4% to 8.04%, respectively.

1. Introduction

Riboflavin (RIB), or vitamin B2 (7,8‐dimethyl‐10‐ribityl‐isoalloxazine), discovered by Warburg and Christie in 1932, is a water‐soluble vitamin essential for numerous metabolic and redox reactions in the human body [1, 2, 3]. Alongside other B vitamins, it contributes to the catabolism of fats and proteins, energy production and antioxidant defence mechanisms [4, 5]. As RIB cannot be synthesized endogenously, it must be acquired from dietary sources including liver, dairy products, whole grains, vegetables, mushrooms and supplementation [6, 7, 8]. Deficiency may lead to neurological issues, photosensitivity, growth retardation, dermatitis and mucocutaneous lesions [9, 10].

Accurate analysis of RIB is important for quality control of fortified foods and supplements, clinical assessment of deficiency and pharmaceutical regulation [11]. Several analytical methods have been reported for RIB determination, including fluorescence [12, 13, 14], chromatography [12, 13, 14, 15, 16], spectrophotometry [17, 18, 19, 20], fluorimetry [21, 22], mass spectrometry [23, 24, 25] and electrochemical techniques [26, 27, 28]. Paper‐based electrochemical sensors have emerged as low‐cost, disposable and biocompatible platforms offering rapid analysis and minimal sample consumption [29, 30]. Their operation relies on capillary transport and reagent immobilization within porous cellulose structures [31, 32, 33, 34, 35].

Nanotechnology integration has significantly advanced the sensitivity and selectivity of paper‐based systems [36]. Nanostructured inks, particularly those incorporating metal nanoparticles, provide high surface area, tunable electrochemical properties and enhanced catalytic activity [37, 38]. Conductive ink‐based electrodes are attractive for biomedical diagnostics, environmental monitoring and point‐of‐care analysis due to their simple fabrication, low cost and robust electrical performance [39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49].

In this study, an innovative disposable electrochemical platform based on paper microelectrodes with the help of novel conductive pen loaded by with silver ink towards quantitative determination of RIB in blood plasma samples. The sensor fabrication process is simple and very fast. Therefore, the sensor preparation is time/cost effective which was significantly reduced.

1.1. Chemicals and Reagents

RIB phosphate, potassium ferricyanide (K₃[Fe (CN)₆]) and potassium ferrocyanide (K₄[Fe(CN)₆]) were obtained from Sigma‐Aldrich (Canada). Sodium hydroxide (NaOH), bovine serum albumin (BSA), silver nitrate (AgNO₃), diacetate alcohol (DAA), polyacrylic acid (PAA) and diethanolamine (DEA) were purchased from Merck (Germany). Human plasma samples were provided by the Iranian Blood Transfusion Research Center (Tabriz, Iran). Deionized water was supplied by Ghazi Pharmaceutical Company (Tabriz, Iran). All reagents were of analytical grade. Silver ink was synthesized according to our previous procedure [44, 45, 46, 47, 48, 49].

1.2. Apparatus

Field emission scanning electron microscopy (FE‐SEM) was performed using a TESCAN system operated at 15 kV. Electrochemical measurements were carried out using a PalmSens 4c potentiostat (Palm Instruments, the Netherlands) controlled by PSTrace software installed on a Lenovo laptop.

1.3. Preparation of Real Samples

Human plasma samples from the Iranian Blood Transfusion Research Centre and stored at −20°C and vortexed daily. For protein precipitation, 0.5 mL of acetonitrile was added to plasma, vortexed for 2 min, and centrifuged at 12,000 rpm for 3 min. A 0.5 mL aliquot of the supernatant was collected and added to the supporting electrolyte.

1.4. Preparation of Conductive Pen

After synthesizing the silver ink, the dispersion was filtered through a 0.2 µm membrane to remove large aggregates. Viscosity was adjusted using 2 wt% hydroxypropyl cellulose. The ink was then loaded into an empty ballpoint pen cartridge to enable direct writing of conductive electrode patterns on cellulose paper. After drying (<1 s), the drawn lines formed stable, conductive pathways suitable for electrochemical sensing (Figure S1, ESI).

2. Results and Discussion

2.1. Electrical, Mechanical and Thermal Stability of Silver Ink on Photographic Paper

The silver‐ink dried immediately after drawing, forming conductive pathways confirmed by LED (light emitting diode) illumination and ohmmeter measurements (Figure S2A, ESI). Mechanical durability was evaluated through repeated bending, rolling and twisting cycles. The sheet resistance remained stable (≈452 Ω) and no cracking or delamination was observed (Figure S2B, ESI). The strong adhesion is attributed to the interlocking of silver ink with cellulose fibres, forming a stable conductive network. Thermal stability was assessed by heating the electrodes to 150°C for 30 s. Resistance remained at 1.5 Ω and the LED remained illuminated (Figure S2C, ESI), indicating minimal thermal degradation. Moisture resistance was confirmed by immersion in water, where the conductivity remained stable (2.9 Ω) (Figure S2D, ESI).

2.2. Fabrication of a Disposable Electrochemical Sensor Using Pen‐On‐Paper Technology

Miniature paper‐based electrodes, specifically the working electrode (WE), reference electrode (RE) and auxiliary electrode (AE), were fabricated on photographic paper using a conductive pen loaded with silver ink. Once the ink dried on the paper surface (in less than a second), 5 µL of RIB was applied onto the sensing area of the electrode, followed by a 20‐min incubation at room temperature. The electrodes were then interfaced with a potentiostat to analyse their electrochemical behaviour through various techniques, including cyclic voltammetry (CV), square‐wave voltammetry (SWV), chronoamperometry (ChA) and differential pulse voltammetry (DPV). During electrochemical testing, WEs included both Ag ink and Ag/RIB ink electrodes. All electrochemical measurements were conducted in a solution containing K₃[Fe(CN)₆]/K₄[Fe(CN)₆] and KCl (0.005 M) mixed in a 1:1 ratio, which served as the supporting electrolyte. The step‐by‐step process of preparing the miniature paper sensor for RIB detection is illustrated in Scheme 1.

SCHEME 1.

Fabrication process of a paper‐based disposable electrochemical sensor for the recognition of RIB. RIB, riboflavin.

2.3. Electrochemical Evaluation of the Designed Sensor and Proposed Mechanism

The electrochemical characterization and functional effectiveness of the sensor for the detection of RIB were systematically evaluated in two separate stages: (I) a pure silver ink‐coated electrode and (II) an RIB immobilized on a silver ink‐based electrode. The characterization was performed using CV, SWV, DPV and ChA, as shown in Figure 1. The bare silver ink electrode stabilized on the surface of paper exhibited remarkable electron transfer behaviour in the Fe(II)/Fe(III) redox system, showing an anodic peak current of approximately 852.4 µA at 0.35 V. After interaction with RIB, a significant increase in the anodic peak current intensity was recorded, reaching 5337.4 µA at the same oxidation potential. The immoblization of RIB caused a higher repulsion of [Fe(CN)₆]^3−/4− on the electrode surface and enhanced the charge transfer. RIB (and its phosphorylated form FMN) is a redox cofactor that can be electrochemically reduced/oxidized via two single‐electron steps (oxidized → semiquinone → hydroquinone). On an electrode, it can act as a surface‐bound redox mediator:

FIGURE 1.

(A–D) CVs, SWVs, DPVs and ChAs of bare Ag‐ink, Ag‐ink/RIB in the presence of ferrocyanide/ferricyanide/KCl (0.005 M) at the potential range of −1 to +1 V and sweep rate of 100 mV/s, respectively. (E–H) Histograms of peak current versus type of electrode obtained from CVs, SWVs, DPVs and ChAs, respectively. RIB, riboflavin.

Electrochemical reduction (on the electrode surface)

$$\mathrm{FMNox}+\mathrm{e}-\rightleftharpoons {\mathrm{FMN}}^{\circ }-\left(\mathrm{semiquinone}\right)$$

$${\mathrm{FMN}}^{\circ }-+\mathrm{e}-\rightleftharpoons \mathrm{FMNred}$$

If FMN is immobilized on the silver surface, it can undergo these redox steps rapidly and then chemically transfer electrons between the electrode and solution redox species (ferri/ferrocyanide). That is, the immobilized FMN can catalyse the ferrocyanide/ferricyanide couple:

Chemical electron transfer (catalytic regeneration)

$$\mathrm{FM}{\mathrm{N}}_{\mathrm{red}}+{\left[\mathrm{Fe}{\left(\mathrm{CN}\right)}_6\right]}^{3-}\to \mathrm{FM}{\mathrm{N}}_{\mathrm{ox}}+{\left[\mathrm{Fe}{\left(\mathrm{CN}\right)}_6\right]}^{4-}$$

Because FMN on the surface is readily oxidised/reduced by the electrode, this cycle regenerates FMN and produces a catalytic current many times larger than the simple diffusion‐limited current of ferri/ferro alone. This explains the large anodic current compared with the Ag‐ink alone.

Table S1 (ESI) provides a comprehensive summary of the experimental data for the detection of RIB using the designed sensor. Comparable results were obtained through SWV, DPV and ChA measurements, which showed peak current responses of 226.5, 45.82 and 35.27 µA, respectively, for the silver ink modified electrode on the paper substrate. After immobilization of RIB molecules on the electrode surface, the peak currents increased to 912.31, 214.56 and 11.99 µA. This increase confirms the sensitivity and efficient interaction between the silver nano‐core and RIB. Moreover, the large distance between the reduction and oxidation peaks and the asymmetry of the current peaks in the forward and reverse scans confirm the irreversibility of the reaction. The strong conductive nature and excellent integration of the silver ink network with the cellulose of paper facilitate rapid electron transfer across the electrode surface. Thus, the silver ink network provides an active electrocatalytic interface that enhances both the response time and the electrochemical signal of the device to RIB oxidation. Silver ink in paper‐based disposable electrochemical sensors can significantly enhance the rate and effective reversibility of electrochemical reactions due to its superior electrical conductivity, high ability to catalyse redox reactions and mechanical stability, making it a highly suitable material for these applications. The CV results show that modifying the electrode surface with silver ink increases the oxidation peak current and reduces the electron transfer resistance, indicating the acceleration of electron exchange between the electrode surface and the target molecules. This confirms the importance of silver ink in improving the performance of electrodes.

2.4. The FE‐SEM and Energy‐Dispersive X‐Ray Spectroscopy (EDS) Characterization of the RIB Sensor

FE‐SEM, EDS and cross‐SEM images were used to systematically investigate the surface morphology, elemental composition and size of the prepared micro‐scale electrodes for the fabrication of a silver ink‐based electrode. Figure 2 shows the FE‐SEM images of the successful formation and immobilization of silver ink on the cellulose micro fibrous network, taken at different magnifications and fields of view. The recorded diameters in different regions of the sample indicate that the particle size range mainly varies between approximately 50 and 75 nm. The mostly homogeneous quasi‐spherical distribution of the nanomaterials and the absence of large pores indicate dense coverage as well as strong interaction between the silver ink and the functional groups of the cellulose. This phenomenon increases the effective surface area and kinetics of electric charge transfer on the substrate and also increases the density of active sites, which is crucial for better electrode performance. Silver conductive inks typically contain metallic Ag nanoparticles (or Ag flakes), surface capping agents/binders and a volatile or semi‐volatile solvent. RIB‐5′‐phosphate (FMN) is an anionic, photoactive molecule with a phosphate group that can coordinate to metal surfaces and participate in redox chemistry when photoexcited. When FMN meets the ink environment, the phosphate moiety and polar parts of FMN will adsorb onto exposed Ag surfaces or displace weakly bound ligands (surface complexation/ligand exchange). This forms an adsorbed layer of FMN/phosphate on Ag. Adsorbed phosphate groups coordinate to the silver surface and can stabilize or neutralize surface charges. This may change the interparticle spacing and affect ink rheology and film formation.

FIGURE 2.

(A and B) FE‐SEM images of microelectrode prepared by Ag nano‐ink before (A) and after incubation with RIB (B) at various magnifications.

SEM cross‐sectional images demonstrate the successful incorporation of RIB immobilized on silver nanoparticle ink onto the porous surface of the paper substrate. Figure 3 shows the penetration depth and adhesion of the deposited layer to the porous network of the cellulose fibre substrate, confirming effective penetration and fixation. The morphology at the electrode‐substrate interface demonstrates strong interfacial bonding and mechanical stability, which are crucial for the durability of the sensor.

FIGURE 3.

Cross FE‐SEM images of microelectrode prepared by Ag nano‐ink before (A) and after incubation with RIB (B) at various magnifications.

According to the quantitative EDX analysis (Figure S3, ESI), before the incubation of RIB, the predominant elements identified in the sample were silver (exhibiting a weight percentage of 98.89 and an intensity of 1495.4) alongside nitrogen (with a weight percentage of 1.11 and an intensity of 10.6). The substantial atomic percentage of silver serves to corroborate the effective modification of the paper substrate facilitated by the ink. After RIB incubation, in addition to silver and nitrogen, the elements carbon, phosphorus and sulphur also appeared with different weight percentages and intensities, with silver (69.47%) still dominant and carbon (9.48%), sulphur (13.56%), phosphorus (3.98%) and nitrogen (3.25%) added to the sample in significant amounts, and the values indicate the validity of the results.

Based on FE‐SEM and EDX results, successful ink stabilization, uniform chemical production and improved structural properties were confirmed which is important to the excellent electronic conductivity of substrate and analytical performance of designed sensor. This uniform elemental distribution is a direct result of the optimized synthesis conditions and plays an important role in ensuring uniform electrochemical performance across the entire sensor surface. This optimized nanostructure provides a high‐density substrate for subsequent immobilization of the bio sensing element RIB, forming a solid foundation for improved analytical performance of the sensor.

2.5. Analytical Performance of RIB's Microscale Disposable Electrochemical Sensor

The analytical capabilities of the engineered miniaturized electrochemical sensor were assessed utilizing the SWV technique. For this purpose, varying concentrations of the analyte (0.2, 0.4, 1, 2, 4, 6, 8 and 10 µM) were applied onto the surface of paper electrodes, and corresponding voltammograms were recorded (Figure 4). As evident from the results (Figure 4B), a linear correlation exists between the sensor's peak current response and the RIB concentration within the linear range of 0.4–10 µM, with the lowest quantifiable concentration established at 0.4 µM. The regression equation obtained, describing the quantitative relationship between the sensor's peak current response and RIB concentration, is as follows: I _p (µA) = 1251.1 C _RIB + 19,341, R ² = 0.9853. These results underscore the miniaturized sensor's potential for accurately determining RIB concentrations.

FIGURE 4.

(A) SWVs of the microsensor using Ag‐ink/RIB electrodes with different concentrations of RIB (0.2, 0.4, 1, 2, 4, 6, 8 and 10 µM) in the scan rate of 10 mV/s. (B) Relationship between I _pa and RIB concentration.

2.6. Bioanalysis of RIB in Human Plasma Samples Using Microscale Paper‐Based Disposable Electrochemical Sensor

The analytical capability of the fabricated paper‐based electrochemical sensor was further validated by analysing of RIB in human plasma samples. Human plasma samples were spiked with different RIB concentrations at a plasma ratio of 1:1 (v/v) to obtain final concentrations in the linear range of 0.4–10 µM. For each measurement, 5 µL of the spiked plasma sample was directly applied to the surface of the engineered Ag‐ink/RIB electrode to incubate under controlled conditions to allow sufficient interaction between RIB molecules and active sites of the WE. The electrochemical responses were recorded by the SWV technique under optimized conditions. The electrochemical behaviour of RIB in blood plasma showed a clear linear relationship between anodic peak current and RIB concentration in the range of interest, confirming the suitability of the sensor for quantitative determination of RIB in biological matrices (R ² > 0.9829).

The calibration curve obtained from the linear regression equation is expressed as I _p (µA) = 1217.5 C _RIB + 8327.4. The lower limit of quantification was 0.4 µM. (Figure S4, ESI).

Table 1 compares previous studies for the determination of RIB in plasma samples using electrochemical methods [50, 51, 52, 53, 54]. Solid substrates (GCE, SPCE) have improved sensitivity and reduced limits of detection (LOD) with graphene, gold or carbon paste electrodes on glass or plastic, as well as surface modification of the electrode with various nanomaterials such as Fe₃O₄NPs, MIPs and AuNPs nanostructures. Although some of these methods have better performance than our sensor (paper‐based electrochemical sensor); however, due to the need for expensive modifier such as gold or composite nanomaterials, these sensors require expensive electrodes, time‐consuming preparation and laboratory equipment. But, designed paper‐based sensors are more disposable and low‐cost (preparation by direct writing by pen‐on‐paper technique).

TABLE 1.

Comparison of the proposed method with other methods for the determination of riboflavin (RIB).

Refs.	LOD/LOQ	Linear range	Technique	Recognition element/material
	LLOQ
[50]	6 nM	0.3–60 µM	SWV	$\alpha$ Fe2o3 NPs/MWCNT/AuNPs/GCE
[51]	1.5 nM	2–40 µM	LSV	2D‐MoS2‐M2O3‐CC/SPCE
[52]	2.38 nM	0.01–0.12 µM	DPV	VB2‐PoAP/MIPs/GCE
[53]	89 nM	0.3–100 µM		Fe3O4 NPs‐rGO/GCE
[54]	250 nM	2–40 µM	SWV	Fe3O4 NPs‐ePADs
[55]	710 nM	1.7–34 µM	CV	Co2+‐Y/CPE
This work	0.2 µM	0.2–10 µM	SWV	Ag‐ink

Abbreviations: CV, cyclic voltammetry; DPV, differential pulse voltammetry; LOD, limits of detection; SWV, square‐wave voltammetry.

3. Analytical Method Validation

3.1. Selectivity

To evaluate the detection power of the micro‐scale sensor, recognition of RIB (1 µM) in the presence of several amino acids, as well as glucose, uric acid, ascorbic acid and dopamine as interfering species, was performed using the SWV technique. Figure S5 (ESI) and Table S2 (ESI) shows the peak current intensity and potential of sensor associated with different interfering agents. As can be seen, the peak current intensity of RIB did not change significantly in the presence of the amino acids AA, Dop, ASP, Gly and glucose, and the slight change in the current peak was negligible. Therefore, the developed disposable electrochemical microscale sensor is suitable for the specific detection of RIB together with these amino acids without any hindrance. Therefore, it is very important to test the selectivity of the proposed microscopic sensor is able recognize of RIB with suitable specificity.

3.2. Stability of the Sensor on Consecutive Applications on Different Days

The daily stability of the microscale sensor was evaluated using the SWV technique in a potential range between −1 and +1 V, and the electrochemical response was monitored for 4 consecutive days. The findings showed that the sensor has suitable stability in the early days, but gradually, with the passage of time and measurement cycles, the recorded current intensity and signal characteristics decreased, which indicates a decrease in the efficiency and stability of the developed sensor (Figure 5). This decrease in peak current intensity could be due to the diffusion of conductive ink on the electrode or physicochemical changes due to repeated measurement effects on the paper substrate, which leads to signal reduction. The gradual decrease in the sensor response over consecutive days is due to the low stability of the conductive structure in the photographic paper substrate. Considering the gradual decline in current intensity over time, it is recommended that the electrode be employed as a disposable component for immediate use, but perhaps not for long‐term storage. This practice prevents possible physicochemical alterations resulting from repeated use and ensures stable and reproducible electrochemical performance.

FIGURE 5.

(A) SWVs of electrochemical micro‐scale sensor in 4 consecutive days. (B) Histogram of peak current of disposable sensor versus storage time.

3.3. Repeatability of the Electrochemical Sensor

Three silver ink‐based paper electrodes were evaluated using the RIB post‐loading method at concentrations of 0.4, 4 and 10 µM in the presence of K₃[Fe(CN)₆]/K₄[Fe(CN)₆] and KCl (0.005 mM), which served as the supporting electrolyte (Figure 6). To evaluate the repeatability of the our disposable paper‐based electrochemical sensor, three same prepared electrodes were analysed under multiple measurements. The calculated standard deviations (SDs) for the three concentrations (0.4, 4 and 10 µM) were 3.4%, 6.1% and 8.04%, respectively. According to FDA guidelines, these values are consistent with recognized standards, RSD_Ave = 5.84 for electrochemical sensors, and confirm the capability of the method to deliver accurate and reliable analytical studies (Table S3, ESI).

FIGURE 6.

(A) Three consecutive SWVs of the three microsensor with three concentrations of RIB (0.4, 4 and 10 µM). (B) Corresponding histogram of peak current versus concentration of analyte in three repeats.

3.4. Reproducibility of the Electrochemical Sensor

To evaluate the reproducibility of the our disposable paper‐based electrochemical sensor, three independently prepared electrodes were analysed under identical conditions at different RIB concentrations, specifically 0.4, 4 and 10 µM (Figure 7). The detection technique used was SWV. Initially, SWVs of the three paper‐based sensors were recorded in a 0.4 µM of RIB. Subsequently, three sensors with a 4 mM of RIB concentration and another three EC sensors with a 10 µM of RIB were sampled under identical conditions. At all concentrations, the obtained peak current intensities were almost overlapping, indicating stable signals and reproducible manufacturing quality among the three microsensors. This consistency indicates that the electrode modification method provides uniform electroactive surface properties and stable electron transfer kinetics across the different batches (Table S4, ESI).

FIGURE 7.

(A) The first, second and third SWVs of the three individual micro sensors with three different concentrations of RIB (0.4, 4 and 10 µM). (B) Corresponding histogram of peak current versus concentration of analyte in three repeats.

Quantitatively, the calculated SD for the RIB current responses at 0.4, 4 and 10 µM concentrations were 6.35%, 1.47% and 4.39%, respectively. These low SD values indicate strong reproducibility confirming that measurement variations are minimal and are mainly attributed to the inherent experimental noise. As a result, the fabricated paper‐based sensors exhibit excellent repeatability and stability confirm the validity of the silver conductive ink‐based microscale sensing platform for the quantitative determination of RIB.

4. Conclusion

In this study we introduced a novel paper‐based micro‐scale electrochemical paper‐based sensor for the rapid and portable detection of RIB, fabricated using pen‐on‐paper technology and electro‐conductive silver ink. Using this sensor RIB was determined in a linear range of 0.4–10 µA, exhibiting appropriate sensitivity and selectivity. For the first time, immediately dryable silver ink was utilized for the rapid and low‐cost fabrication of disposable electrochemical sensors which is key item of this report and opens new perspectives in the development of cost‐effective, accurate, low‐cost and disposable electrochemical sensors for clinical diagnostics. The platform can be further advanced through improved materials and fabrication strategies, supporting rapid biomarker analysis in challenging samples such as human plasma. These findings led to further progress in the design of paper‐based microsensors for electrochemical bioanalysis.

Author Contributions

Rokhsareh Ebrahimi: writing – original draft, methodology, investigation. Mohammad Hasanzadeh: conceptualization, supervision, writing – review and editing, validation, formal analysis, methodology. Nasrin Shadjou: writing – review and editing, project administration.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: ansa70069‐sup‐0001‐SuppMat.docx.

Data Availability Statement

Data available on request due to privacy/ethical restrictions.